New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group.
Bioconjug Chem
; 28(6): 1741-1748, 2017 06 21.
Article
en En
| MEDLINE
| ID: mdl-28485576
ABSTRACT
Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.
Texto completo:
1
Colección:
01-internacional
Asunto principal:
ADN
/
Resonancia Magnética Nuclear Biomolecular
/
Elementos de la Serie de los Lantanoides
Idioma:
En
Revista:
Bioconjug Chem
Asunto de la revista:
BIOQUIMICA
Año:
2017
Tipo del documento:
Article
País de afiliación:
Australia